Matrix-associated Laser Desorption/ionization Time-of-flight Research Articles

570. Impact of a Blood Culture Direct Identification Procedure on Time to Identification for Gram-Negative Organisms and Antimicrobial Management in Bloodstream Infections

Abstract Background Multiplex polymerase chain reaction (PCR) and microarray diagnostic methods for rapid identification (ID) in Gram-negative bloodstream infections (GNBSI) remain unavailable in many healthcare facilities. Use of other methods such as matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) along with rapid sample preparation from positive blood cultures, may decrease time to ID and targeted antimicrobial therapy in BSI. This study compares time to ID and final susceptibilities of Gram-negative bacteria (GNB) in positive blood cultures using a rapid MALDI-TOF direct ID process versus conventional culture methods, as well as impact on antimicrobial therapy in GNBSI. Methods This retrospective cohort included patients ≥18 years of age who had a positive blood culture with a GNB from January 1 through December 31, 2022, at a hospital serviced by the microbiology lab at Banner University Medical Center Phoenix (conventional ID) or Banner University Medical Center Tucson (direct ID). Direct ID consisted of using MALDI-TOF for immediate ID of a centrifuged sample from a GNB-positive blood culture bottle. The primary outcome was time to GNB ID between the two groups. Secondary outcomes included time from positive blood culture to susceptibility reporting, definitive antibiotic regimen, and hospital discharge. Statistical analysis performed with Wilcoxon Rank-Sum testing for continuous data, and Chi square testing for nominal data. Results A total of 254 patients were evaluated, with 100 included in each group. Median time to bacterial ID in the direct group vs. conventional group was 6.1 hr vs 29.2 hr (P< 0.0001). No differences in secondary outcomes, including times to final susceptibility (54.0 hr vs 60.9 hr, P=0.73), definitive antibiotic regimen (49.5 hr vs 44.9 hr, P=0.42), and hospital discharge (132.5 hr vs. 124.4 hr, P=0.85) were noted, though this study may have been unable to detect differences in such outcomes due to variance in organism resistance patterns and clinical practice between facilities. Conclusion Direct MALDI-TOF ID significantly decreases time to organism identification in GNBSI compared with conventional methods. Utilization of direct ID plus antimicrobial stewardship interventions in GNBSI is an area of opportunity. Disclosures All Authors: No reported disclosures

Structural Characterization of Glycerophosphorylated and Succinylated Cyclic β-(1→2)-d-Glucan Produced by Sinorhizobium mliloti 1021.

Rhizobia produces different types of surface polysaccharides. Among them, cyclic β-(1→2)-d-glucan is located in the periplasmic space of rhizobia and plays an important role in the adaptation of bacteria to osmotic adaptation. Cyclic β-(1→2)-d-glucan (CG), synthesized from Sinorhiozbbium meliloti 1021, has a neutral and anionic form. In the present study, we characterized the exact chemical structures of anionic CG after purification using size exclusion s (Bio-Gel P-6 and P-2) chromatography, and DEAE-Sephadex anion exchange chromatography. The exact structure of each isolated anionic CG was characterized using various analytical methods such as nuclear magnetic resonance (NMR), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and matrix associated laser desorption ionization-time of Flight (MALDI-TOF) mass spectrometry. The precise chemical structures of novel anionic CG molecules were elucidated by various NMR spectroscopic analyses, including 1H, 13C, 31P, and 2D HSQC NMR spectroscopy. As a result, we discovered that anionic CG molecules have either glycerophosphoryl or succinyl residues at C6 positions of a neutral CG. In addition, the results of MALDI-TOF mass spectrometric analysis confirmed that there are two types of patterns for anionic CG peaks, where one type of peak was the succinylated CG (SCG) and the other was glycerophospholated CG (GCG). In addition, it was revealed that each anionic CG has one to four substituents of the succinyl group of SCG and glycerophosphoryl group of GCG, respectively. Anionic CG could have potential as a cyclic polysaccharide for drug delivery systems and a chiral separator based on the complexation with basic target molecules.

Avaliação da especificidade do ágar Violeta Vermelho Bile Glicose para o isolamento de Enterobacteriaceae em leite de cabra cru

Introduction: Coliforms have been used over the years as indicator microorganisms for microbiological quality. However, several studies report the lack of direct correlation between their presence and that of pathogens in the evaluation of food safety, leading to the suggestion of new analysis proposals, such as the search for total enterobacteria, which includes all members of the Enterobacteriaceae family. Objective: This study aimed to determine the population of enterobacteria in raw goat milk samples based on the microbiological criteria suggested by the National Health Surveillance Agency - Ministry of Health (Anvisa - MS) - Public Consultation Noo 542 of July 17, 2018 for Pasteurized milk samples. Method: 21 samples from small producers from different regions of the state of Rio de Janeiro were used. Isolates were obtained from inoculation on VRBG agar according to ISO 21528-2 and identified by Matrix Associated Laser Desorption-Ionization – Time of Flight (MALDI/TOF) mass spectrometry. Results: Of the 222 isolates identified, only 26.6% belonged to the Enterobacteriaceae family. Another 61.3% belonged to the Pseudomonas genus, 4.5% to the Acinetobacter genus and 7.6% were Stenotrophomonas maltophilia. Although there are no microbiological parameters in Anvisa-MS RDC No 12 of January 2, 2001 for raw milk, the results of isolation on VBRG agar led to the investigation of other studies with similar findings regarding isolation of VRBG bacteria from goat and bovine milk samples. Conclusions: Detection in VRBG agar of non-Enterobacteriaceae-negative oxidase bacteria, such as those of the genus Acinetobacter, suggests the need for a debate on the methodology indicated by ISO 21528-2 for enumeration of enterobacteria, as the new regulation on standard microbiological data for food will no longer use the coliform group, but the Enterobacteriaceae/g or mL count, as an indicator.

Short-term culture for rapid identification of anaerobic bacteria from blood cultures

Identification of anaerobic bacteria causing blood stream infections (BSI) is challenging. This study describes the epidemiology of anaerobic BSI at a tertiary care hospital and the performance of a rapid method for identification of anaerobic bacteria from blood cultures over three years, between June 2015 and June 2018. Short-term culturing is a low-cost user-friendly method and may be used for rapid identification of bacteria from positive blood cultures by matrix-associated laser desorption ionization time of flight (MALDI-TOF). Short-term culturing is performed after Gram staining on all positive blood culture bottles (BCBs) before 10:30 a.m. in our laboratory. Successful short-term cultures were defined as growth and reliable MALDI-TOF result in maximum 8 h after culture positivity. Data pertaining to unique anaerobic episodes and short-term cultures was collected retrospectively from our laboratory information system. Overall, during the three-year period, 692 unique anaerobic episodes (including Propionibacterium spp) were isolated. A total of 17 anaerobic bacterial genera were isolated in our laboratory, with 5 GNB genera and 12 GPB genera. The most prevalent bacteria were Bacteroides spp. 266/692 (38%), Propionibacterium spp. 128/692 (18%), Clostridium spp. 103/692 (15%), Fusobacterium spp. 34/692 (5%), and Actinomyces spp. 34/692 (5%). We performed short-term cultures on 270/564 (48%) clinically relevant episodes (excluding Propionibacterium spp) on chocolate agar plates. Growth within 8 h from culture positivity was detected in 33/270 (12%) short-term cultures. There were 22/33 (67%) gram negative (GNB) and 11/33 (33%) gram positive bacteria (GPB). Only two genera were identified: Bacteroides spp 22/33 (67%) and Clostridium spp 11/33 (33%). Thus, short-term culturing can function as a low-cost add-on to already existing protocols involving MALDI-TOF, where both GNB and GPB can be identified.

Epidemiology and Resistance Patterns of Bacterial and Fungal Colonization of Biliary Plastic Stents: A Prospective Cohort Study.

BackgroundPlastic stents used for the treatment of biliary obstruction will become occluded over time due to microbial colonization and formation of biofilms. Treatment of stent-associated cholangitis is often not effective because of inappropriate use of antimicrobial agents or antimicrobial resistance. We aimed to assess the current bacterial and fungal etiology of stent-associated biofilms, with particular emphasis on antimicrobial resistance.MethodsPatients with biliary strictures requiring endoscopic stent placement were prospectively enrolled. After the retrieval of stents, biofilms were disrupted by sonication, microorganisms were cultured, and isolates were identified by matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and/or biochemical typing. Finally, minimum inhibitory concentrations (MICs) were determined for various antimicrobial agents. Selected stents were further analyzed by fluorescence in situ hybridization (FISH).ResultsAmong 120 patients (62.5% males, median age 64 years) with biliary strictures (35% malignant, 65% benign), 113 double pigtail polyurethane and 100 straight polyethylene stents were analyzed after a median indwelling time of 63 days (range, 1–1274 days). The stent occlusion rate was 11.5% and 13%, respectively, being associated with a significantly increased risk of cholangitis (38.5% vs. 9.1%, P<0.001). Ninety-five different bacterial and 13 fungal species were detected; polymicrobial colonization predominated (95.8% vs. 4.2%, P<0.001). Enterococci (79.3%), Enterobacteriaceae (73.7%), and Candida spp. (55.9%) were the leading pathogens. Candida species were more frequent in patients previously receiving prolonged antibiotic therapy (63% vs. 46.7%, P = 0.023). Vancomycin-resistant enterococci accounted for 13.7%, extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae with co-resistance to ciprofloxacin accounted for 13.9%, and azole-resistant Candida spp. accounted for 32.9% of the respective isolates.ConclusionsEnterococci and Candida species play an important role in the microbial colonization of biliary stents. Therefore, empirical antimicrobial treatment of stent-associated cholangitis should be guided toward enterococci, Enterobacteriaceae, streptococci, anaerobes, and Candida. To determine causative pathogens, an accurate microbiological analysis of the extracted stent(s) may be helpful.

Proteomic identification of potential Clonorchis sinensis excretory/secretory products capable of binding and activating human hepatic stellate cells.

Epidemiological and experimental evidence demonstrated that Clonorchis sinensis is an important risk factor of hepatic fibrosis and cholangiocarcinoma. C. sinensis excretory/secretory products (CsESPs) are protein complex including proteases, antioxidant enzymes, and metabolic enzymes, which may contribute to pathogenesis of liver fluke-associated hepatobiliary diseases. However, potential CsESP candidates involved into hepatic fibrosis and cholangiocarcinoma still remain to be elucidated. In the present study, we performed proteomic identification of CsESP candidates capable of binding and activating human hepatic stellate cell line LX-2. Immunofluorescence analysis confirmed the interaction of CsESPs with LX-2 cell membrane. LX-2 cells could be stimulated by CsESPs from 24 h post incubation (p < 0.05). Specifically, 50 μg/ml of CsESPs showed the strongest effect on cell proliferation in methyl thiazolyl tetrazolium (MTT) assay which could also be demonstrated by flow cytometry analysis (p < 0.01). Furthermore, expression level of human type III collagen in LX-2 cells treated with CsESPs was significantly higher than that in control cells measured by molecular beacon and semiquantitative reverse transcription (RT)-PCR approaches (p < 0.01). Finally, CsESPs before and after incubation with LX-2 cells were subjected to two-dimensional gel electrophoresis (2-DE) analysis and matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Nine proteins with abundance change above threefold were Rho GTPase-activating protein, mitochondrial cytochrome c oxidase subunit Va, α-enolase, phospholipase C, interleukin-15, insect-derived growth factor, cytochrome c oxidase subunit VI, DNAH1 protein, and kinesin light chain. Taken together, we identified potential CsESP candidates capable of binding and activating human hepatic stellate cells, providing more direct evidences that are previously unknown to accelerate strategies for C. sinensis prevention.

Characterization of cAMP Response Element-Like Sequence in Gonadal Specific Aromatase Promoter Sequence ofXenopusEmbryos

The p450 aromatase gene has a tissue-specific promoter that is regulated by specific transcriptional factors. In rats and humans, a cAMP response element-like sequence (CLS) and an NR5A1/NR5A2 binding sequence have been identified as cis elements in the aromatase promoter; these cis elements mediate cAMP-induced expression in the ovaries and testes. CLS is recognized by a cAMP-responsive element binding protein (CREB) as the principal component. In this study, we performed a gel shift assay to analyze the proteins that interact with the cis-element in Xenopus aromatase. An electrophpretic mobility gel shift assay (EMSA) and matrix-associated laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis of the proteins responsible for retarding the mobility of CLS revealed that ATF4 interacted in vitro with CLS in gonadal specific aromatase promoter sequence of Xenopus embryos. Although a significant difference was observed in aromatase mRNA expression between male and female gonads, no difference in the expression of ATF4 was observed between them at stage 50. With regard to aromatase expression in the gonad of Xenopus embryos, ATF4 might act in combination with multiple transcription factors as a trans-element of CLS in place of CREB.

Species-specific identification and differentiation of Arcobacter, Helicobacter and Campylobacter by full-spectral matrix-associated laser desorption/ionization time of flight mass spectrometry analysis

Rapid and reliable identification of Arcobacter and Helicobacter species, and their distinction from phenotypically similar Campylobacter species, has become increasingly important, since many of them are now recognized as human and/or animal pathogens. Matrix-associated laser desorption/ionization-time of flight (MALDI-TOF) MS has been shown to be a rapid and sensitive method for characterization of micro-organisms. In this study, we therefore established a reference database of selected Arcobacter, Helicobacter and Campylobacter species for MALDI-TOF MS identification. Besides the species with significance as food-borne pathogens - Arcobacter butzleri, Helicobacter pullorum, Campylobacter jejuni and Campylobacter coli - several other members of these genera were included in the reference library to determine the species specificity of the designed MALDI Biotyper reference database library. Strains that made up the reference database library were grown on Columbia agar, and yielded reproducible and unique mass spectra profiles, which were compared with the Bruker Biotyper database, version 2. The database was used to identify 144 clinical isolates using whole spectral profiles. Furthermore, reproducibility of MALDI-TOF MS results was evaluated with respect to age and/or storage of bacteria and different growth media. It was found that correct identification could be obtained even if the bacteria were stored at room temperature or at 4 degrees C up to 9 days before being tested. In addition, bacteria were correctly identified when grown on Campylosel agar; however, they were not when grown on modified charcoal cefoperazone deoxycholate agar. These results indicate that MALDI-TOF MS fingerprinting is a fast and reliable method for the identification of Arcobacter and Helicobacter species, and their distinction from phenotypically similar Campylobacter species, with applications in clinical diagnostics.

Comparative Analysis of the Tear Protein Expression After Photorefractive Keratectomy Using Two-Dimensional Electrophoresis

Purpose: To investigate the change in the tear protein composition of patients who underwent refractive surgery. Methods: Tear samples were collected before photorefrative keratectomy (PRK), on the first, the second, and the third postoperative day, and then a month after the operation from 40 eyes of 20 patients. These tear samples were analyzed using two-dimensional gel electrophoresis (2-DE). Matrix-associated laser desorption/ionization time of flight (MALDI-TOF) was employed for the identification of expressed proteins. Control tear samples were collected from 40 eyes of 20 healthy volunteers who had no history of ocular surgery or pathology. Results: On the first postoperative day, lipocalin-1 precursor, lipocalin-1, and lysozyme were up-regulated. On the second postoperative day, serum albumin precursor and serum albumin were up-regulated. The tears collected on the third postoperative day and after 1 month had similar protein expression levels to the control group. Lipocalin 1 precursor and lysozyme were up-regulated and down-regulated after reftactive surgery, respectively. However, each protein had a different molecular weight and isopotential point. Conclusions: The tear protein composition changed uniquely in the early postoperative period, and proteins with different isopotential points were detected after PRK. We hypothesized that the healing process might influence the expression of the tear proteins.

Identification by proteome analysis of muscle proteins in sea bream (Sparus aurata)

The present paper reported postmortem changes in muscle proteins on sea bream (Sparus aurata), during ice storage. The postmortem evolution of protein patterns in farmed sea bream was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimension electrophoresis (2DE). Matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was used for identification of proteins. Results of the bio-informatics analysis demonstrate that: (1) the normalized volume of skeletal alpha-actin and tropomyosin is not affected by the time of storage; (2) the normalized volumes of myosin light chain 3 and of major histocompatibility complex (MHC) class II beta1 increase; (3) the normalized volumes of Sec 13-like and parvalbumin significantly decrease.

Insulin-like growth factor-I downregulates embryonic myosin heavy chain (eMyHC) in myoblast nuclei

The obscure ability of the insulin-like growth factors (IGF-I & -II) to stimulate both myoblast proliferation and differentiation suggests that the latter effect may be mediated locally, possibly by IGF binding proteins (IGFBPs). In some cells, the growth inhibitory actions of IGFBP-5 require plasma membrane translocation and nuclear localization. Immunoreactivity of presumably endogenous IGFBP-5 was identified within proliferating rat L6 myoblast nuclei using fluorescent and confocal microscopy in separate experiments and was reduced by 100 ng/ml IGF-I in a time-dependent manner. Western blotting of nuclear and cytosolic protein identified a single anti-IGFBP-5 immunoreactive protein of approximately 200 kDa, primarily in nuclear fractions, that was downregulated in cells treated with IGF-I for 12 h. The unknown protein was immunopurified from nuclear fractions and identified as the rat homologue for embryonic myosin heavy chain (eMyHC) using matrix-associated laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy. Cross-reactivity of the IGFBP-5 antiserum with eMyHC was confirmed by blotting anti-IGFBP-5 nuclear immunoprecipitates with eMyHC monoclonal antibodies (F1.652). These data indicate that eMyHC is located predominantly within the nuclei of proliferating L6 myoblasts and suggest that IGF-stimulated differentiation is associated with the rapid downregulation of nuclear eMyHC as these cells stop expressing this myosin II isoform as they differentiate. Myosin Iβ has been identified within the nuclei of non-muscle cells where it helps to regulate gene transcription. Thus, eMyHC may serve a similar role in myoblasts that is specific only to the undifferentiated state.

Plasmodium berghei ookinetes bind to Anopheles gambiae and Drosophila melanogaster annexins

Using a proteomic approach we identified polypeptides from Anopheles gambiae and Drosophila melanogaster protein extracts that selectively bind purified Plasmodium berghei ookinetes in vitro; these were two and three distinct polypeptides, respectively, with an apparent molecular weight of about 36 kDa. Combining two-dimensional electrophoresis and MALDI-TOF (matrix-associated laser desorption ionization time of flight) mass spectrometry we determined that the polypeptides correspond to isomorphs of the annexin B11 protein of the fruit fly. When protein extracts derived from A. gambiae and D. melanogaster tissue culture cells were further fractionated, the binding activity matching the annexin protein could be localized in the fraction derived from cell membranes in both diptera. Antibody staining showed that annexin also binds to ookinetes during the invasion of the mosquito midgut. Finally, inclusion of antiannexin antisera in a mosquito blood meal impaired parasite development, suggesting a facilitating role for annexins in the infection of the mosquito by Plasmodium.

Effect of Cryopreservation on Sea Bass Sperm Proteins

In the present study we used two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to verify whether the protein expression of sea bass sperm was affected by the cryopreservation procedure. The protein profiles differed between fresh and frozen-thawed semen as revealed by visual inspection and by image analysis software. We identified 163 spots in fresh sperm; among these, 13 were significantly decreased and 8 were absent in two-dimensional gel obtained with cryopreserved sperm. Five of these spots were analyzed with MALDI-TOF, but only three showed a significant match in the databases used in bio-informatics analysis (PeptIdent, Mascot, and MS-Fit). In particular, spot 5 showed homology with a novel protein of zebrafish (similar to SKB1 of human and mouse), spot 13 showed homology with amphibian G1/S-specific cyclin E2, and spot 20 showed homology with the hypothetical protein DKFZp566A1524 of Brachidanio rerio. The present work shows that the use of the cryopreservation procedure causes the degradation of sperm proteins and among these, two could be at least partially responsible for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm.

Mechanisms of Innate and Adaptive Immune Responsiveness to Bacterial Infection in Cornea

Purpose. The dysfunctional tear syndrome of Sjogren’s syndrome (SjS) is associated with focal CD4+ cell infiltration of the lacrimal glands and serum autoantibodies to intracellular proteins. To learn how lacrimal acinar cells (AC) expose or present autoantigen epitopes, we wish to identify autoantigens in amenable experimental models. Methods. Subcellular fractions from rat lacrimal glands were analyzed by SDS-PAGE. Western blots were probed with sera from NOD mice with diagnosed SjS-like disease, and IgG1 heavy chain was detected with isotype specific secondary antibody. Labeled bands were analyzed using matrix-associated laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry. Tryptic peptides were identified by Mascot Matrix Science search software. Compartmental distributions of candidate B cell autoantigens were determined by Western blotting of isolated membrane fractions from rabbit AC cultured with and without chronic carbachol (CCh) stimulation. Results. Subcellular fractionation analysis indicated that the candidate autoantigens were primarily associated with the membrane phase; most were primarily in compartments of the biosynthetic and lysosomal pathways. Some also were in secretory vesicle-related compartments of control cells but re-distributed to the endosomes after chronic CCh stimulation. The proteomic analysis identified Grp 78, rab3D, and ribosomal protein L7 as candidate autoantigens. Western blotting with monoclonal antibodies confirmed the presence and compartmental distributions of Grp 78 and rab3D. Protein sequence analysis with TEPITOPE confirmed that an identified B cell epitope at the N-terminal end of L7 also is a likely T cell epitope. Conclusion Grp78 and L7 are known to be antigens in other autoimmune diseases but have not previously been known to be associated with SjS. Rab3D has not previously been recognized as a disease-associated autoantigen. These and other AC autoantigens are present in intracellular compartments where they may be subject to aberrant proteolytic processing that we theorize generates otherwise cryptic epitopes. Commercial Relationship(s): None; Support: EY13720, EY05801, DK48522

Removal of sodium and potassium adducts using a matrix additive during matrix‐associated laser desorption/ionization time‐of‐flight mass spectrometric analysis of peptides

Monovalent cations often associate with peptides and proteins under mass spectrometry (MS) conditions, resulting in a discernable, but often misleading, adduct cluster pattern. These adduct cluster peaks reduce the signal intensity of specific peptide species by splitting the ion population into multiple mass peaks, suppressing the ionization of neighboring low-abundance peaks, and interfering with identification of post-translational modifications. Further, monovalent contaminants tend to form a distribution of matrix cluster peaks in matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) spectra causing interference and suppression in the mass range below 1400 Da. The most common method for reduction or elimination of adduct clusters is solid-phase extraction via a pipette tip or spin column, which often leads to loss of low-abundance peptide components. In this study we describe the use of a commercially available surfactant blend that markedly reduces the adduction of monovalent cations during peptide analysis by MALDI-TOFMS.